siRNA represents a promising therapeutic technology, however hurdles remain to be overcome in the administration of siRNA to a patient. Various techniques have been employed to introduce siRNA into cells including cationic lipid-mediated transfection, viral delivery and nucleofection. Delivery using cationic lipids appears to be the easiest method for transfection, however its efficiency is often low and limited to specific types of cells. While viral delivery works well for some difficult cells, cell lines, and for in vivo delivery (Garraway et al., 2007, J Pharmacol Exp Ther. 322:982-8) it has raised safety concerns. Unmodified siRNA single strands or duplexes are extremely unstable in serum, with a half-life of less than 1 minute for the single strands and 3.3 to 5 minutes for the duplexes (Sioud, 2005. Curr Drug Targets. 6:647-53; Sioud and Iversen, 2005. Curr Drug Targets. 6:647-53). However, chemical modifications of the siRNAs have demonstrated dramatic improvement in stabilities; modified siRNA were approximately 900-fold more stable than unmodified siRNAs (Morrissey et al., 2005. Nat Biotechnol. 23:1002-7). Certain stable and active siRNA compounds are disclosed in PCT patent publications WO 2008/050329 and WO 2009/044392, assigned to an applicant of the present invention and hereby incorporated by reference in their entirety.
Delivery of siRNAs to the central nervous system (CNS) is problematic because of the blood brain barrier (BBB) and most successful strategies have used an intrathecal route. Intracerebroventricular infusion of naked siRNA at a high concentration via minipumps into the rodent brain have successfully inhibited target gene expression, however, knockdown of target gene expression drops off significantly in regions distant from the infusion site (Thakker et al., 2004. Proc Natl Acad Sci USA. 101:17270-5). Using a lumbar indwelling cannula attached to an osmotic minipump, intrathecal application of high doses (400 μg/day) of siRNAs in the spinal cord silenced the ATP-gated cation-channel P2X3, and relieved chronic neuropathic pain (Dorn et al., 2004. Nucleic Acids Res. 32:e49). Other studies achieved therapeutic effects after intrathecal delivery with substantially lower doses using an implanted Alzet pump in a chronic mouse model for ALS progression (Wang et al., 2008. J Biol Chem. 283:15845-52) and with pretreatment in acute pain models (Christoph et al., 2006. Biochem Biophys Res Commun. 350:238-43; Luo et al., 2005. Mol Pain. 1:29). Rohl and Kerreck review various methods of RNAi in pain models (J. Neurochem. 99:371-380). Inoue has implicated RhoA activation in neuropathic pain (Inoue et al., 2004. Nat Med 10(7): 712-8).
Contusive injuries to the spinal cord produce primary damage to the tissue and initiate a cascade of events called secondary injury, which progresses in several phases over days to weeks (Beattie et al., 2000. J Neurotrauma. 17:915-25). Microglia within the spinal cord become activated rapidly after injury. Activated macrophages begin to accumulate by 3 days after spinal cord injury in the rat (Schwab et al., 2004. Glia. 47:377-86). During the first several days after SCI there is extensive cell death in and around the injury site and the axonal processes of neurons die back from the injury site. These processes are accompanied by complex molecular changes including upregulation of RhoA and its activation in both neural (D'Alessandri et al., 1995. Curr. Eye Res. 14:911-926; Dubreuil et al., 2003. J Cell Biol. 162:233-43; Erschbamer et al., 2005. J Comp Neurol. 484:224-33; Yune et al., 2007. J Neurosci. 27:7751-61) and immune cells (Fu et al., 2007. J Neurosci. 27:4154-64; Pixley et al., 2005 J Cell Sci. 118:1873-83; Schwab et al., 2004), which are believed to contribute to secondary damage as well as to limit axonal regeneration and functional recovery. Cell permeable Rho antagonists (e.g. BA-210), which inhibit the activity of phosphorylated Rho family proteins including RhoA, have shown improved recovery after spinal cord injury in rodents (Lord-Fontaine et al., 2008. J Neurotrauma. 25:1309-22) and are being tested in clinical trials to promote axon regeneration and functional recovery (Baptiste et al., 2009. Expert Opin Investig Drugs. 18:663-73). RhoA protein levels are upregulated several fold reaching their highest levels in the rat spinal cord ˜7 days following spinal cord contusion, providing a window for therapeutic intervention after SCI (Erschbamer et al., 2005 supra). However, BA-210 inhibition of RhoA was found only up to 4 days after acute treatment but not at one week (Lord-Fontaine et al., 2008) when RhoA expression is robust (Erschbamer et al., 2005). Methods for targeted delivery of siRNA to the CNS and enhanced activity in the target cells would be desirable.
Pain is the most common symptom of disease and the most frequent complaint with which patients present to physicians. Inadequate pain management remains a major public health problem. Currently available treatment options for allodynia/SCI associated pain are limited and since many SCI pain conditions are not amenable to treatment, particularly at-level and below-level neuropathic pain, management becomes largely symptomatic. A solution for the treatment of allodynia/SCI associated pain is therefore required.